Bacterial transglycosylases are enzymes that couple the disaccharide subunits of peptidoglycan to form long carbohydrate chains. These enzymes are the target of the pentasaccharide antibiotic moenomycin as well as the proposed target of certain glycopeptides that overcome vancomycin resistance. Because bacterial transglycosylases are difficult enzymes to study, it has not previously been possible to evaluate how moenomycin inhibits them or to determine whether glycopeptide analogues directly target them. We have identified transglycosylase assay conditions that enable kinetic analysis of inhibitors and have examined the inhibition of Escherichia coli penicillin-binding protein 1b (PBP1b) by moenomycin as well as by various glycopeptides. We report that chlorobiphenyl vancomycin analogues that are incapable of binding substrates nevertheless inhibit E. coli PBP1b, which shows that these compounds interact directly with the enzyme. These findings support the hypothesis that chlorobiphenyl vancomycin derivatives overcome vanA resistance by targeting bacterial transglycosylases. We have also found that moenomycin is not competitive with respect to the lipid II substrate of PBP1b, as has long been believed. With the development of suitable methods to evaluate bacterial transglycosylases, it is now possible to probe the mechanism of action of some potentially very important antibiotics.

Moenomycin A is the only known natural product that inhibits peptidoglycan biosynthesis by binding the bacterial transglycosylases. We describe a degradation/reconstruction route to manipulate the reducing end of moenomycin A. A comparision of the biological and enzyme inhibitory activity of moenomycin and an analog containing a nerol lipid in place of the natural C 25 lipid chain provides insight into the role of the moenocinol unit. Our results show that a lipid chain having ten carbons in moenocinol is sufficient for enzyme inhibition, but a longer chain is required for biological acitivity, apparently because the molecule must partition into biological membranes to reach its target in bacterial cells.Moenomycin A (1, Scheme 1) is a natural product that inhibits peptidoglycan biosynthesis by binding to the bacterial transglycosylases (TGases). 1 On a molar basis, moenomycin is a thousand times more potent than vancomycin, but poor pharmacokinetic properties related to the C25 isoprenoid chain have prevented its use in humans. 2 Removing this unit completely abolishes biological activity. 3 Whether this portion of the molecule can be replaced by a shorter lipid is unclear for two reasons: first, until now there have been no methods to remove the natural lipid chain and replace it with other lipids without also altering other structural features of the molecule; 4 second, assays to evaluate the TGase activity of moenomycin and derivatives in the absence of biological membranes 5 have only recently become available 6 , making it difficult to dissect the contribution of the isoprenoid chain to enzyme binding versus membrane anchoring 7 . Here we describe a degradation/ reconstruction route to manipulate the reducing end of moenomycin. We evaluate the enzyme inhibitory activity of moenomycin and an analog containing a nerol chain against kahne@chemistry.harvard.edu. Supporting Information Available: Experimental procedures and spectral data for all compounds. Over-expression and purification conditions for E. faecalis PBP2a are also described. This material is available free of charge via the Internet at http://pubs.acs.org. We faced three challenges in developing a degradation/reconstruction route to moenomycin A. First, although there has been considerable work on the degradation of moenomycin A, 8 a synthetic route to degrade 1 to the intact pentasaccharide 3 has not been reported. Second, we needed to develop a synthesis of the 2-O-moenocinyl glycerate (11, Scheme 2). Model systems for the lipid glycerate have been synthesized previously, but the chemistry could not be extended to the natural lipid. 9 Third, we needed an efficient method to form the phosphoglycerate linkage to 3. NIH Public AccessPrevious studies on the degradation of moenomycin A have shown that under protic acid conditions the glycosidic bonds of the pentasaccharide core begin to decompose before the anomeric phosphate bond is cleaved. 8a We envisioned a solution to this problem that takes advantage of the known lability of allyl et...

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The glycopeptide antibiotics prevent maturation of the bacterial cell wall by binding to the terminal d-alanyl-d-alanine moiety of peptidoglycan precursors, thereby inhibiting the enzymes involved in the final stages of peptidoglycan synthesis. However, there are significant differences in the biological activity of particular glycopeptide derivatives that are not related to their affinity for d-Ala-d-Ala. We compare the ability of vancomycin and a set of clinically relevant glycopeptides to inhibit Staphylococcus aureus PBP2 (penicillin binding protein), the major transglycosylase in a clinically relevant pathogen, S. aureus. We report experiments suggesting that activity differences between glycopeptides against this organism reflect a combination of substrate binding and secondary interactions with key enzymes involved in peptidoglycan synthesis.